Current Status of White Spot Disease (Caused by

WSSV) & its Effect in Shrimp Culture

WSSV - White Spot Syndrome Virus

White Spot Syndrome Virus,

Its Many Names: HHNV- hypodermal & hematopoietic necrosis

baculovirus (China 1993-94). CBV - China baculovirus (China ~1994). SEMBV/ Red Disease - systemic ectodermal &

mesodermal baculovirus (Thailand, India 1994) . PRDV / PAV - penaeid rod-shaped DNA virus /

penaeid acute viremia (Japan 1995). WSBV - white spot baculovirus (Taiwan 1996). WSBV - white spot bacilliform virus (China 2001) WSV - white spot virus (OIE 2000). WSSV - white spot syndrome virus (Taiwan 1996). WSSV - accepted by ICTV as type species in

Nimaviridae: Whispovirus (8th Report ICTV, 2005).

Recent WSD Outbreaks

2012: Brunei – in Penaeus stylirostris

2012: Madagascar – in P. monodon 2011: Saudi Arabia – in P. indicus 2011: Mozambique – in P. monodon Other countries already WSSV

infected: A few with WSSV-free compartments Most continue to manage around

WSD.

THE WHITE SPOT VIRUS PANDEMIC(Year of First Occurrence by Location)

19921993

1993

19941994

1995

1999/2011

19951996/1997

12/1999

9/19991/1999

19972000

2001

2002/2005

2005

2004

20052011

2011 2012

Biology of the virus (WSSV)

IHH

NV

- 20

nm

HPV

- 22

nm

BP, MBV, BMN ~75 x 300 nm

WSSV ~130 x 350 nm

The Main DNA Viruses of Penaeid Shrimp

Scale Bar divisions are 20 nm

From: Lo, C.F., Aoki, T., Bonami, J.R., Flegel, T., Lightner, D.V., Walker, P. J. and Vlak, J.M.. 2012.” Family Nimaviridae, Genus Whispovirus. 8ht Report of the ICTV:Virus Taxonomy. pp. 229-234.

White Spot Syndrome Virus*Family Nimaviridae n.f.; Genus Whispovirus; Type Species: WSSVReplication Nuclear

Morphology enveloped, elliptical rod with prominent apical "tail"

Size virion: 80-120 x 250-380 nm nucleocapsid: 58-67x230-350 nm

Density 1.18-1.25 g/ml

Nuclei Acid dsDNA, circular, supercoiled, ~305 kb

ORFs ~531 ORFs for 181 functional proteins

* Various publications 1995-2012

From: Virus Taxonomy; 9th Report of the International Committee on Taxonomy of Viruses, 2012

In terms of virulence, one major WSSV strain appears to have caused the global pandemic.

Strain(s) with lower virulence recently reported.

Genome sequencing information shows that there are numerous, apparently minor, genetic variants.

WSSV- multiple strains?

New genotypes of WSSV from the Kingdom of Saudi Arabia, Mozambique and Madagascar

WSSV genome

ORF94

ORF125

CGCAAAAAGCGTGCCGCACCTCCACCTGAGGATGAAGAAGAGGATGATTTCTAC

AG/TAACAAGGAGGAAGAAGACGCGAGGATCAAGCGTGCAGTCGACATGGCTGTTGCAGCCATCAACGAAA

54-bp RU

69-bp RU

Two variable tandem repeat regions within ORF94 and ORF125 were used to genotype WSSV samples. RU: repeat unit.

Map of Saudi Arabia showing the location of shrimp farms (A-D) that were infected with WSSV.

Persian Gulf

Farm D

Farm A

Iraq Iran

WSSV

Yemem

Red Sea

Jeddah

Jazan

Eritrea

Saudi Arabia

Farm B

Farm C

WSSV

WSSV

WSSV

Number of repeat units (RUs) found within ORF125 & ORF94 of WSSV isolated from KSA farms, Mozambique (MZ) &

Madagascar (MD)Genotype a No. RUs in

ORF125 No. of RUs in ORF94

KSA Farm site

Isolate no.

{6125, 794} 6 7 A 10-143{8125, 1394} 8 13 B 11-065/6{8125, 1394} 8 13 B 11-065/7{8125, 1394} 8 13 B 11-102/1{8125, 1394} 8 13 B 11-102/2{8125, 1394} 8 13 B 11-114{7125, del94} 7 del b C 11-041/20{7125, del94} 7 del C 11-041/21{7125, del94} 7 del C 11-041/22{7125, del94} 7 del C 11-208/3{7125, del94} 7 del C 11-208/4{7125, del94} 7 del C 11-208/8{7125, del94} 7 del C 11-208/15{7125, del94} 7 del D 11-394/A{7125, del94} 7 del D 11-394/B{6125, del94} 6 del MZ 11-312{7125, del94} 7 del MD 12-171a: The genotypes are categorized as {N125, N94} where N is the number of repeat units in a specific ORF indicated by the subscript.b: deletion

PCR primers and PCR profiles used in WSSV genotying analysis

ORF PCR primer Sequence PCR condition ReferenceORF94 ORF94-F 5’-

TCTACTCGAGGAGGTGACGAC

94oC, 5 min

35 cycles of94oC/30 s,55oC/30 s,72oC/1-1.5 min

72oC, 7 min

Wongteerasupaya et al. 2003. Dis Aquat Org 54: 253-257.ORF94-R 5’-

AGCAGGTGTGTACACATTTCATG

ORF93-F1 5’-CGCCCTATTACCATTGATGC

Tang et al. 2012.Dis Aquat Org

ORF96-R1 5’-GCAACAAATTCCCCTTTCAA

ORF125 ORF125-flank-F 5’-CGAAATCTTGATATGTTGTGC

Dieu et al. 2004. J Gen Virol 85: 3607-3618.

ORF125-flank-R 5’-CCATATCCATTGCCCTTCTC

12.0 kb 2.0 kb 1.0 kb 650 bp 400 bp

M 1 2 3 4 5 6 7 8 M 1 2 3 4 5 6 7 8

A B

Farm A B B B C C D MZ Farm A B B B C C D MZ

10-1

43

11-0

65

11-1

02

11-1

14

11-0

41

11-2

08

11-3

94

11-3

12

10-1

43

11-0

65

11-1

02

11-1

14

11-0

41

11-2

08

11-3

94

11-3

12

6-R

U

13-R

U

13-R

U

13-R

U

WSSV genotying in the ORF94 region. (A) PCR with primers ORF94-F/R; (B) PCR using primers ORF93-F1/ORF96-R1. WSSV isolated from Saudi Arabia’s farms (A to D) and from Mozambique (MZ); PCR products (marked by a circle) from lane 2 and lane 6, respectively, were purified and sequenced, their sequences are aligned as shown in below.

UAZ isolate #

CGCCCTATTACCATTGATGCACAAATTTCCTCCTTCATTCTAATAGCGGCAGATTGTTTGTCAAAATAACACTCCCTATAGTAACAACCAGGATTTC----∆----AATGCGTGGGCAGAGGCGGAGGTGGTGATAAAGCGTTTCTGAGAAACATTGGGCGTATGACGTCAACTACATTATTCTTCCTCCTCCTCCTCCTCCTATTGCCTCTGCCAGTTCAGTATTTTATTTTTCTTCCTATACAATAAAAAGTATCAGATGAATATTTTTACTGTTTTTCTGTTCATCCCTCTTTCCTATTGTAAAAAAACCAATAACTAACTAATCATGGATAACTTGAAAGGGGAATTTGTTGC

A PCR fragment amplified from primers, ORF93F1 and ORF96R1, showing deletions (---∆---) in ORFs 94, 95 and 93. Nucleotide sequence of the 348-bp amplicons from WSSV sample no. 11-208/3 (see Fig above, lane 6); letters in bold are the sequence of primers.

ORF93 ORF96ORF94 with 13-RU ORF95

Schematic alignment of a WSSV isolate no. 11-208/3 (Fig B above, lane 6) to the isolate no. 11-065/6 (lane 2), which consists of 13-RU within ORF94. (---): represents a 1,502-bp deletion; (■): PCR primers ORF93-F1 and ORF96-R1.

WSSV isolate # 11-208/3

WSSV isolate # 11-065/6 (normal, pandemic form)

Three genotypes: {6125, 794}, {7125, del94} , and {8125, 1394} were found from 15 Saudi Arabia WSSV isolates.

One genotype of {6125, del94} was found in Mozambique.

Another genotype {7125, del94} was found in Madagascar.

The WSSV genotypes of {7125, del94} and {6125, del94} appear to be new variants with a 1,522-bp deletion encompassing complete regions of ORF94 and ORF95 and the first 82-bp of ORF93.

Conclusions:

WSSV Natural and Experimental Hosts

List of known natural & experimental hosts for WSSV is 104 species (all are decapods, except artemia):

Penaeid shrimps & prawns Freshwater prawns Crabs - several genera Spiny lobsters Freshwater crayfish susceptible genera:

North American, European & Australian Artemia spp. – passive carrier - does not become

infected

Pathology of White Spot Disease

White Spot/Red Disease

Clinical Signs Sudden reduction in feeding, lethargic. Red discoloration in P. monodon, P. vannamei

& P. stylirostris. Soft, loose shells. White spots 0.5 to 2 mm under cuticle (less

common in Western Hemisphere penaeids). Up to 100% mortality within 3 days of onset

of disease signs.

WSSV in P. monodon, Madagascar, April 2012Photo courtesy of Marc LeGroumellec, UNIMA

Aquamen Farm, South Madagascar, April 2012

Case 2000-15; Penaeus vannamei

WSSV in Penaeus vannamei – Ecuador 1999photo courtesy of Diego Buenaventrua

WSSV in Penaeus monodon: India & Thailand

WSD Santa Catarina,P. vannamei, Brazil 2005

WSSV in Penaeus vannamei – Ecuador, 1999

DIAGNOSTIC & DETECTION METHODS

MethodDirect BF microscopyPhase microscopyDarkfield microscopyHistopathologyBioassay/histologyTransmission EM /SEMAntibody-based w/PAb, MAb

DNA Probes - ISH

YHV-group+

+/?-

++++++

++

+++

TSV++++

++++++

+++

+++

WSSV

Detection by PCR/RT-PCR +++ +++

June 2012

IHHNV+--

+++ +++

+/?

++++++

---

+++ +/-+-

++++++

DNA Probes - Dot Blot +++ - - +++

Stomach P. chinensis

Stomach P. chinensis

Stomach P. chinensis

Stomach P. vannamei

HEO

Gills LO

Antennalgland

H&E - Uropod

ISH / DNA Probe

H&E - stomach

ISH / DNA Probe

UAZ Case 99-326 (P. stylirostris)WSSV Mab 8B7F6 (Immuno-Squash with whole fixed gills)

Nested PCR: WSSV

Lo’s nested-941 bpLN4 1st step-732 bp

Lo’s 1st step-1447 bp

LN4 nested-668 bp

1st step Nested

The Asian Pandemic

1993

1991/2

1993

19931993

1994

1999

Photo courtesy of Dr. Barry Hill, CEFAS, UK

Introduction from Asia & Spread of WSSV in the

Americas

(date) = eradicated from affected farms

(1995)

1999

(1997)

(1996)

1999

1999

19992000

1999

2007

Route of WSSV introduction

into the Americas WSSV most likely reached the Americas

in IQF or block frozen shrimp that were re-processed at costal plants.

Solid waste from reprocessing was dumped into penaeid & crab nursery areas exposing wild penaeids & other decapods to WSSV.

Wild shrimp in the Gulf of Mexico & Pacific side of Panama were the first found to be WSSV +.

BIOLOGY & HISTORY OF WSSV IN THE AMERICAS

1995: first seen in Texas in farmed juvenile L. setiferus.

1995-98: found in imported frozen shrimp. 1997 & 98: found in wild penaeids in Texas & SC. 1996 & 97: found in freshwater crayfish at National

Zoo. 1997 & 98: in SC farm cumulative mortalities >99%

in L. stylirostris & L. vannamei. January 1999: found in wild & farmed L. vannamei &

L. stylirostris in Central America w/ severe losses. June 1999: found in Ecuador w/ severe losses. December 1999: reached Mexico. January 2005: confirmed in Brazil. February 2007: detected in Louisiana crayfish farms.

Mechanisms for WSSV International Transfer &

Introduction

Imported Live PLs or Broodstock

Asia Middle East Turkey & Greece Central &

S.America (after introduction)

ImportedFrozen Shrimp

United States Central

America Australia Spain

WSSV in Frozen Imported Commodity Shrimp from

AsiaSurveys of US & Australian markets found high prevalence of WSSV+ commodity shrimp imported from SE Asia:1995-96:USA - 50% – Nunan, Poulos & Lightner,

1998, Aquaculture.1998-99: USA - 50% – Durand, Tang & Lightner,

2000. J Aquatic Animal Health.2003: USA - 73% – Montgommery-Brock & Tamura,

2005, Industry Briefs, USMSFP. 2004: Australia – 38% - East et al. 2004, DAFF2005: USA - 4.7% (0% to 38.7%) – Reville et al. J

Shellfish Research2006: USA – 10% - Hasson et al. 2006, Aquaculture

America.2007: Australia – 100%- 2007, Aust. Prawn Farmers

Assoc.

P. monodon; UAZ 95-204

Whole tail: 1.1 x107 WSSVWhole head: 2.5 x10 7 WSSV

Peeled shells (shell, tail fan & pleopods): 4.76 x108 WSSV

Peeled tail muscle plus gut: 3.4 x107 WSSV

WSSV copy number/g DNA in shrimp with acute phase WSSV infection

Current Management Strategies for White Spot

Disease in the Americas

Increased biosecuritySource water treatment to exclude wild

vectorsReduced water use & higher culture

densitiesSPF (WSSV-free) shrimp stocks Use SPR lines of shrimpHyperthermia & WSSV

Reduction of farming in cold seasonUse of greenhouses

Water Temperature is one environmental factor with a profound effect on WSSV replication &, hence, on expression of White Spot

Disease in infected populations.

Hyperthermia and White Spot Disease

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 20 40

Days Post Initial Exposure

Num

ber S

urvi

ving

Injected / 32 C

Injected / 26 C

Per os / 32 C

Per os / 26 C

Inj conrol saline / 26C

Inoculum / 33C/1hr

Effect of hyperthermia on WSD in L. vannamei

Modified from Vidal et al. 2001

Controlled environment farms near Tumbes, Peru

Controlled environment farms near Tumbes, Peru (Jan. 2005)

Covered ponds in Guangzhou Province, China

Covered ponds in Guangzhou Province, China

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

WSSV

Effect of WSSV on Chinese shrimp aquaculture

Effect of WSSV on Ecuador’s Shrimp

Exports

Source: Panorama Acuicola, 2001, 6(3):27

0

10

20

30

40

50

60

70

$Million

USA Europe Asia Canada

199819992000

ECONOMIC IMPACT OF WHITE SPOT DISEASE OF SHRIMP

Ecuador (1999) US$ >280 million lost Ecuador (2000) US$ >575 million lost

All farms affected. Production area of 175,000 hectares halved. >500,000 jobs lost. Presidential decree of State of emergency issued. National Stability Fund used.

World Production of ShrimpCapture Fisheries & Aquaculture

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008

Million MT

Capture Fisheries Aquaculture Source: FAO (2011).Source GOAL 2011; Notes: M. rosenbergii is not included; China includes freshwater production of P. vannamei.

26 %

28%

52%Global viral

pandemics:1992 to ~2002

**

ESTIMATED ECONOMIC LOSSES FROM DISCOVERY

TO PRESENT VIRUS SINCE/YEAR PRODUCT LOSS

IHHNV- Americas* 1981 $ 0.5-1 billion YHV - Asia 1991 $ 0.5 billionTSV-Americas 1991/92 $ 1-2 billionTSV-Americas 1999 $ 0.5-1 billionWSSV - Asia 1992/93 $ > 6 billion WSSV - Americas 1999 $ 1-2 billionIMNV – Americas 2004 $ 100-200 millionIMNV – Indonesia 2006 $ 1 billion??

* Includes Gulf of California fishery 1989-1994

Thank you for your attention!